REL 2024-11-29 2025-09-25 2025-09-25 0000-0001-9724-9547 Rene Frank Astbury Centre, School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds Willow Terrace Road Leeds United Kingdom LS29JT 0000-0001-9724-9547 Rene Frank Astbury Centre, School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds Willow Terrace Road Leeds United Kingdom LS29JT 0000-0001-8560-7407 Julia Peukes Chan Zuckerberg Imaging Institute Redwood City United States CA 94065 0000-0003-3990-6910 John A. G. Briggs Max Plank Institute of Biochemistry Martinsried Germany 82152 Planegg 0000-0002-7171-0494 Charlie Lovatt Rosalind Franklin Institute Didcot United Kingdom OX11 0QS Peukes J Lovatt C Leistner C Boulanger J Morado DR Fuller MJG Kukulski W Zhu F Komiyama NH Briggs JAG Grant SGN Frank RAW European Research Council (ERC) ERC-CoG-648432 MEMBRANEFUSION Germany Medical Research Council (MRC, United Kingdom) MC_UP_1201/16 United Kingdom UK Research and Innovation (UKRI) MR/V022644/1 United Kingdom Biotechnology and Biological Sciences Research Council (BBSRC) BB/M011151/1 United Kingdom Wellcome Trust SBF005/1046 United Kingdom Wellcome Trust 108466/Z/15/Z United Kingdom Wellcome Trust 208395/Z/17/Z United Kingdom 442.9 10.6019/EMPIAR-12795 EMDB tissue EMD-53528 Peukes J Lovatt C Leistner C Boulanger J Morado DR Fuller MJG Kukulski W Zhu F Komiyama NH Briggs JAG Grant SGN Frank RAW eLife 10.7554/eLife.100335.1 In-tissue (vitreous cryo-section) synapse tilt series /data/in-tissue_synapse_data/tiltseries_files tilt series MRC MRC 50 1 32 BIT FLOAT variable variable variable variable

In-tissue (vitreous cryo-section) synapse reconstructed volumes (4x binned) /data/in-tissue_synapse_data/tomogram_files reconstructed volumes MRC MRC 50 1 32 BIT FLOAT variable variable variable variable

data/segmentations/in-tissue_synapse_fig2.cxs ChimeraX session file containing segmentation of in-tissue (vitreous cryo-section) synapse (related to Fig. 2). Ultrafresh (synaptoneurosome) synapse tilt series /data/ultrafresh_synaptoneurosome_data/tiltseries tilt series MRC MRC 93 1 32 BIT FLOAT variable 2.984 variable 2.984

Ultrafresh (synaptoneurosome) synapse reconstructed volumes (4x binned) /data/ultrafresh_synaptoneurosome_data/tomograms reconstructed volumes MRC MRC 93 1 32 BIT FLOAT variable 11.2 variable 11.2

data/segmentations/ultrafresh_synapse_segmentations_motl.em Motive list (.em) containing coordinates of PreSM and PoSM segmentations of ultrafresh (synaptoneurosome) synapses: 1: Cross-correlation coefficient 2: Dataset number 3: Tomogram number 4: running particle number 5: Running number of tomogram - used for wedge list 6: Index of feature in tomogram 7: Running tomogram number 8: X-coordinate in full tomogram 9: Y-coordinate in full tomogram 10: Z-coordinate in full tomogram 11: X-shift in subvolume - AFTER rotation of template 12: Y-shift in subvolume - AFTER rotation of template 13: Z-shift in subvolume - AFTER rotation of template 14: X-shift in subvolume - BEFORE rotation of template 15: Y-shift in subvolume - BEFORE rotation of template 16: Z-shift in subvolume - BEFORE rotation of template 17: Phi 18: Psi 19: Theta 20: Class number data/segmentations/ultrafresh_synapse_fig1.cxs ChimeraX session file containing segmentation of ultrafresh (synaptoneurosome) synapse (related to Fig. 1).